The present study aims to elucidate the role of the apolipoprotein E gene (APOE) in Alzheimer's disease (AD) from a novel epigenetic perspective. The proposed experiments will determine if a causal connection exists between epigenetic characteristics of the APOE 4 allele and the biological alterations responsible for AD. The 4 allele of APOE is the most recognized genetic risk factor for AD. Mainstream research into the role of APOE in AD has primarily focused on the apoE protein and isoform-specific differences in its structure and function. That approach has generated broad and valuable insights and hypotheses. However, the precise mechanism by which the apoE4 exerts its detrimental effect in AD remains obscure. For example, the apoE4 protein has clear detrimental effects on the central nervous system, yet these effects do not fully account for the age-related aspects of AD onset and progression. Over the years, independent studies have suggested that other biological effects of APOE, besides the apoE protein isoforms, may contribute to overall AD risk. My laboratory has been studying the APOE gene for the past five years in an attempt to identify additional functional elements that might contribute such novel biological effects to AD. Recently, we have identified an excellent candidate in the 4 allele-carrying exon of APOE. Our preliminary studies show that this exon is imbedded in an authentic CpG island (CGI) that is highly methylated in human postmortem brain (PMB), and exhibits transcriptional enhancer activity. Our work provides evidence that this APOE CGI can differentially modulate expression of genes at the APOE locus in a cell type-, DNA methylation- (DNAm), and 4 allele- specific manner. Currently, it is unknown if this CGI and DNAm of APOE have any critical biological functions or consequences that impact AD risk. Based on this evidence, we hypothesize that the 4 allele of APOE is a critical element of the APOE CGI, which is epigenetically marked, modulates the expression of multiple genes at the APOE locus, and exerts a synergistic effect with its protein product to increase a person's risk for AD. The short-term goal of this work is to assess the epigenotype-phenotype correlations between the APOE CGI and the risk of AD, and to determine if DNAm of the APOE CGI is an integral element of the strong association signal between the 4 allele and the risk of AD. The specific aims of our study will investigate several components of this overall paradigm. Aim 1 will examine the DNAm profile of the APOE CGI, in various tissues of PMB, to identify 4 allele- and AD disease- specific differentially methylated regions. Such differences in DNAm have great potential to serve as a new class of AD biomarkers, in which monitoring epigenetic alterations might indicate disease progression. Aim 2 will inspect the effects of DNAm on the enhancer activity of the APOE CGI. Correlations between DNAm levels of the APOE CGI and RNA expression levels of AD-associated genes will be evaluated. Additionally, the results will be stratified by the 2/3/4 alleles of the APOE CGI to determine the specific effects of the 4 allele. Aim 3 will explore the regulatory mechanisms and protein binding properties of the APOE CGI to determine its functional core region and identify relevant binding proteins, which will provide a more comprehensive view of the 4 allele's role in AD risk. A number of key gaps exist in our current understanding of the link between the APOE 4 allele and AD risk; filling these gaps forms the basis for this proposal. The studies proposed herein will define a new paradigm of APOE gene action in AD. Results from this project will also provide fundamental new knowledge regarding the nature of epigenetic influence and regulation in AD. In turn, this work should provide crucial insights for new strategies of intervention and prevention for AD.